Nitric oxide (NO) is one of the key regulatory molecules produced by endothelial nitric oxide synthase (eNOS, a constitutive isoform of nitric oxide synthase produced in endothelial cells), which plays a significant role in vascular homeostasis. NO counteracts pro-proliferating agents and growth factors, inhibits platelet aggregation, leukocyte adhesion, and maintains vascular smooth muscle cells (VSMCs) in a quiescent state. Vascular injury that leads to denudation of endothelium such as due to balloon inflation and/or stenting leads to the loss of endothelial cells producing vasoprotective eNOS, thereby leading to vascular deficiency of NO at the injured site. Studies have demonstrated an inverse relation between endothelial integrity and VSMC proliferation. Fishman et al., Lab Invest., 32:339-51 (1975).
Treatment modalities for vascular injury focus either on inhibiting VSMC proliferation or on promoting endothelial regeneration; however, an ideal anti-restenotic therapy should be able to achieve both of these effects as well as prevent constrictive remodeling of the artery. Endothelialization is key to the long-term patency of the artery, which is often delayed or inhibited in stented artery, and is implicated as one of the main causes of rebound of restenosis or fatal thrombosis. Finn et al., Arterioscler Thromb Vasc Biol., 27:1500-10 (2007). Thus, augmenting vasoprotective enzyme-eNOS and its “sustained” presence in the injured vessel wall is expected to maintain the vasoprotective functions of endothelium till re-endothelialization takes place via natural mechanisms. Though augmentation of NO levels in the injured vessel wall is a potential therapeutic strategy, its use is limited because of short half-life and high reactivity. The use of NO donors (e.g., organic nitrates, nitroglycerin, etc.) and NO-adducts with other pharmacological agents (e.g., NO-aspirin, NO-releasing statin derivatives etc.) is further limited due to the tolerance and potential hypotensive adverse effects. Napoli et al., Annu Rev Pharmacol Toxicol., 43:97-123 (2003). Localized eNOS gene transfer using HVJ-liposomes (von der Leyen et al., Proc Natl Acad Sci USA., 92:1137-41 (1995)) and adenovirus has shown to limit intimal hyperplasia (Janssens et al., Circulation., 97:1274-81 (1998); Varenne et al., Circulation, 98:919-26 (1998)); however, adenovirus carries the risk of inflammatory response and systemic expression of the transgene. Herz J, Gerard R D.; Proc Natl Acad Sci USA., 90:2812-6 (1993). Biodegradable nanoparticles (NPs) loaded with recombinant eNOS protein could potentially be a better alternative to eNOS gene transfer as NPs can release the encapsulated eNOS protein in active form for a prolonged period of time, irrespective of the functional nature of cellular machinery, which may be compromised under diseased conditions, thus influencing the efficiency of gene expression. Further, depending upon the response, the dose and duration of protein delivery in the target artery can be modulated with NPs, which is limited with gene therapy approaches.